Eur. J. Biochem. 102, 615-623 (1979)
Regulation of Ca2+Efflux from Kidney and Liver Mitochondria by Unsaturated Fatty Acids and Na' Ions Izabela ROMAN, Piotr GMAJ, Cecylia NOWICKA, and Stefan ANGIELSKI Department of Clinical Biochemistry, Institute of Pathology, Medical Academy, Gdai7sk (Received January 30/August 28, 1979)
The effects of fatty acids and monovalent cations on the Ca2+efflux from isolated liver and kidney mitochondria were investigated by means of electrode techniques. It was shown that unsaturated fatty acids and saturated fatty acids of medium chain length (CIZ and C I ~ induced ) a Ca2+ efflux from mitochondria which was not inhibited by ruthenium red, but was specifically inhibited by Na' and Li'. The Ca2+-releasingactivity of unsaturated fatty acids did not correlate with their uncoupling activity. In kidney mitochondria a spontaneous, temperature-dependent Ca2 efflux was observed which was inhibited either by albumin or by Na'. It is suggested that the net Ca2+ accumulation by mitochondria depends on the operation of independent pump and leak pathways. The pump is driven by the membrane potential and can be inhibited by ruthenium red, the leak depends on the presence of unsaturated fatty acids and is inhibited by Na' and Li'. It is suggested that the unsaturated fatty acids produced by mitochondrial phospholipase A2 can be essential in the regulation of the Ca2+ retention in and the Ca2+ release from the mitochondria. +
The role of mitochondria in the maintenance of the steady state Ca2' concentration in kidney cells is well documented [1,2]. However, of the two processes involved in net calcium transport, namely the Ca2+ uptake and the Ca2+ release, only the former had been subjected to thorough investigations [3,4]. It is now generally accepted that the driving force for the mitochondrial Ca2+ uptake is provided by the electric field generated across the inner mitochondrjal membrane by respiration or ATP hydrolysis [5,6], and that the process is catalysed by a specific Ca2+-binding glycoprotein located in the inner mitochondrial membrane and inhibited by ruthenium red and lanthanides [7-91. The Ca2+ transport was reconstituted by the incorporation of the isolated mitochondrial Ca2+-binding glycoprotein into artificial lipid bilayer membranes, whereby its role in Ca2+ transport was confirmed [lo, 111. The stoichiometry of charge transfer accompanying the Ca2 uptake was a matter of some debate. Thus, direct measurements under conditions of limited Ca2+ loading provided the n value of 2 [ 5 , 6 ] ,but deviations from the Nernstian Ca2' distribution were commonly encountered in measurements of the Ca2+ accumulation [12]. A problem of independent pathways for +
Abbreviations. ClPhzC(CN)*, carbonylcyanide m-chlorophenylhydrazone; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; EGTA, [ethylenebis(oxoethylenenitrilo)]tetraaceticacid.
the Ca2+ uptake and release had thus been put forward [13,14]. In the mitochondria from heart and from other excitable tissues a specific Na"/Ca2 antiporter was identified which might operate as a Ca2+-releasing mechanism [15-171. This antiporter is absent in the mitochondria from kidney and liver, and thus the pathways of the Ca2+ efflux from these mitochondria remain to be identified. The retention of accumulated Ca2+ in mitochondria provides a closely related problem. Normally, in the absence of ATP, Ca2+ is retained in the isolated mitochondria for a brief period of time only, and subsequently it is released into the suspending medium in an auto-accelerating process [13,18]. The Ca2+ retention and release depends on several seemingly unrelated external factors such as the Ca2+ to protein ratio, the ionic composition of the medium, the presence or absence of phosphate, the oxidation/ reduction of pyridine nucleotides, and others [12,18, 191. The basic mechanisms which control the Ca2+ retention, however, are unknown. In this paper the data are presented which indicate that the endogenous or exogenous unsaturated fatty acids and saturated fatty acids of medium chain length may be involved in the catalysis of CaZ+ 'leak' from mitochondria by a mechanism partly independent of their uncoupling activity. The Ca2+ efflux induced by unsaturated fatty acids is specifically inhibited +
Fatty-Acid-Induced C a 2 + Efflux from Mitochondria
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by Na', what may provide an important regulatory link in the intracellular Ca2+ homeostasis.
Mitochondria
MATERIALS AND METHODS Mitochondria were isolated from the liver and from the kidney cortex of female Wistar rats by a conventional differential centrifugation procedure. The tissue was disrupted in a Potter homogenizer with a teflon pestle in a medium containing 0.25 M sucrose, 0.01 M Tris-C1 buffer pH 7.4 and 0.1 mM EGTA. The mitochondrial pellets were washed three times. In the last two washes no EGTA was used. The mitochondrial protein was measured by a biuret method with bovine serum albumin as standard. The standard incubation medium in a final volume of 2.5 ml contained: 120 mM KCI or NaCl (see legends), 5 mM Hepes-Tris buffer pH 7.2, 0.5 mM MgC12, 3 mM Tris-succinate, 1 pM rotenone and 1 mg mitochondrial protein per ml. CaC12 was added for either 0.1 mM or 0.5 mM final concentration (limited loading and massive loading conditions, respectively). Fatty acids were added as freshly prepared ethanolic solutions, kept at 0 ° C until used. Other additions were (see legends for the figures): bovine serum albumin (essentially fdtty-acid-free, Sigma Chem. Co., St Louis, Mo., U.S.A.) 5 mg/ml; Na-ATP 3 mM; ruthenium red (Sigma) 1 p M ; CIPhzC(CN)2 (Calbiochem) 0.5 pM. Medium Ca2+ was continuously monitored with a Ca2+-sensitive electrode (F-2112, Radiometer, Denmark) connected to a pH-meter, antilogarithmic amplifier and recorder. H movements and oxygen uptake were measured simultaneously with a glass and a Clark-type oxygen electrodes, respectively. All electrodes were mounted in the same closed, thermostatted reaction chamber. The responses of the Ca2+ and H electrodes were calibrated before each measurement. To simplify the drawings, H + and O2 traces are shown only when relevant information has been provided. Mitochondria1 swelling was measured by recording the changes in absorbance of mitochondrial suspensions at 520 nm in a Zeiss-Opton recording spectrophotometer. All reagents used were of analytical grade. +
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RESULTS Fjfects of Temperature on Energy-Dependent Uptake, Retention and Release o j Cu2+by Kidney Mitochondria
In the energy-linked uptake, the Ca2+ distribution between the mitochondria and the suspending medium reaches a steady state which presumably is maintained by the equilibrium between the Ca2+ uptake
Fig. 1. Temperature dependence of rhe Ca" uptake and release by kidney mitochondria. Incubation medium contained: 120 m M KC1, 0.5 mM MgC12, 5 mM Hepes-Tris pH 7.2, 3 mM Tris-succinate. 1 pM rotenone and 100 pM CaC12. The Ca2+ uptake was started by addition of kidney mitochondria 1 mg/ml
and release. Fig. 1 shows that the steady state of Ca2+ distribution and the retention of Ca2+ by kidney mitochondria are powerfully influenced by temperature. At 20 "C practically all Ca2+ added (100 nmol/mg protein) was taken up and retained until oxygen had been exhausted, i.e. for at least 8 min. As the temperature was raised a progressively smaller proportion of the total Ca2+ present was taken up and, after a period of steady-state maintenance which was the shorter the higher the temperature had been, a spontaneous release of the accumulated Ca2+ was clearly seen. Sharp upper deflection points in Fig. 1 correspond to the exhaustion of oxygen in the suspending medium. Apparently, the inside negative membrane potential disappears during anaerobiosis, what results in a rapid and complete release of the accumulated Ca2+.The initial rate of the Ca2+ uptake, on the other hand, was much less dependent on temperature than the Ca2+ release. This might suggest that the Ca2+ uptake and efflux are catalysed by systems with different temperature dependence. The temperaturedependent Ca2+ release from liver mitochondria, albeit observable, was much less pronounced than that from kidney mitochondria. This discrepancy will be discussed later on. Eflects o f Sodium and Bovine Serum Albumin
Fig.2 shows that the usual, spontaneous Ca" release from kidney mitochondria incubated at 30 "C can be abolished if K + is substituted with N a + as the main cation in the medium. In NaCl medium, the amount of Ca2+ taken up was considerably greater, the period of the steady-state retention having been prolonged, and the efflux phase much less pronounced. When bovine serum albumin (essentially fatty-acidfree) was added to the medium, the spontaneous CaZ+ release disappeared completely : the mitochondria took up all the Ca2+ added and retained it until oxygen had been exhausted. In the presence of bovine
I. Roman, P. Gmaj, C. Nowicka, and S. Angielski
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Fig.2. Eflects of' Na' ions and albumin on the Ca2' uptake and releasc by kidney mitochondria. Experimental conditions as in Fig. I . ( ~--) KCI medium; (----) 120 m M NaCl was substituted for KCI in the medium. Albumin concentration 5 mg/ml. Incubation temperature 30 C
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serum albumin, Na+ ions were of no effect on both Ca2 uptake and release. These experiments allowed for the following conclusions: (a) the spontaneous temperature-dependent Ca2 release from kidney mitochondria depends on the presence of some factor removable by bovine serum albumin, probably fatty acids, and (b) N a + ions inhibit the fatty-acid-dependent Ca2+ efflux, but have no effect on the Ca2+ uptake, having been ineffective in the presence of bovine serum albumin. Therefore, in further experiments the effects of exogenous fatty acids and N a + ions on the Ca2+ release were investigated in more detail. +
+
E f f k t s of' Exogenous Linolenic Acid and Sodium Ions on Cu2+Elflux born Kidney and Liver Mitochondriu In Fig. 3 the effects of exogenous linolenic acid on the Ca2+ release from liver and kidney mitochondria are shown. The experiments were performed at 25 "C to avoid a large spontaneous Ca2 release. Linolenate as added after the Ca2+ uptake had been completed, evoked an immediate, graded Ca2+ release from both liver (Fig. 3A) and kidney (Fig. 3B) mitochondria. A few minutes after linolenate had been added, a new steady state was established at a much lower Ca2+ accumulation level. Upon exhaustion of oxygen from the medium the residual mitochondria1 Ca2 was immediately and completely released. In both liver and kidney mitochondria the Ca2 efflux induced by linolenate was inhibited by Na+ ions. Thus, the addition of linolenate at 25 "C closely mimicked the spontaneous Ca2 release observed in kidney mitochondria at higher temperatures (compare Fig.2 and 3). In +
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Fig. 3. Efiects of' linolenic acid (C18 3 ) arid No+ ions on rhr Ca2 release .from liver ( A ) and kidney ( B ) mitochondria. Incubation KCI medium, (-----) NaCl medium. conditions as in Fig. 1. (-) Linolenic acid added as ethanolic solutions in concentrations: liver mitochondria -20 nmol/mg protein, kidney mitochondria - 10 nmol/mg protein. Incubation temperature 25 C +
liver, as compared to kidney mitochondria, the concentration of linolenate necessary to evoke Ca2 release was approximately twice as high. A lower sensitivity to exogenous linolenate and a lower spontaneous Ca2 release suggest that the endogenous fatty acid level in liver mitochondria is low. (Direct measurements have shown that the levels of endogenous free fatty acids are 3 to 10 times higher in kidney than in liver mitochondria, depending on the fatty acid species: unpublished results of P. Gmaj.) In kidney mitochondria, on the other hand, the effects of endogenous and exogenous fatty acids may sum up producing a higher rate of the Ca2+ efflux. The bottom panels of F i g 3 show that the mitochondrial respiration is activated upon an addition of linolenate. This is the well known uncoupling activity of unsaturated fatty acid [20,21]. However, the stimulation of respiration is identical in both NaCl and KCI media. Thus, the Ca2+ release induced by linolenate cannot be attributed solely to its uncoupling activity and to the collapse of the membrane potential: an inhibition of the Ca2+ efflux by N a + indicates a specific effect of linolenate on the Ca2+ transport. This conclusion had been substantiated by the experiment shown in Fig.4. The Ca2+ efflux from kidney mitochondria induced by a classical uncoupler CIPhzC(CN)2 was not influenced by N a +
+
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Fatty-Acid-Induced Ca2+ Efflux from Mitochondria
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to the presence of ATP or Pi, since it was drastically decreased upon lowering of the Ca2+ to 0.1 pmol/mg protein (not shown). As in previous experiments, sodium ions strongly inhibited the linolenate-induced Ca2+efflux. In kidney mitochondria, on the contrary, the sensitivity to exogenous linolenate was decreased in the presence of ATP, and higher linolenate concentrations were necessary to induce Ca2+ release than those under limited loading conditions. The interpretation of these results are not quite clear yet, but they suggest that the rate of the linolenate-induced Ca2+ efflux from liver mitochondria depends on the amount of Ca2 accumulated in the mitochondrial matrix: when it is increased, the Ca2+ efflux rate at a given level of linolenate becomes increased, too. In kidney mitochondria, the added linolenate may possibly be metabolished in ATP-dependent reaction(s). This organ specificity requires further investigations.
ions. In the latter case, the CaL+release might indeed result directly from the collapse of the membrane potential. Efects o j Linolenute in the Presence o f A T P and Pi The effects of linolenate on the Ca2+ efflux from kidney and liver mitochondria were further investigated under 'massive loading' conditions (0.5 pmol Ca2+/mg protein) in the presence of ATP and inorganic phosphate. The results are shown in Fig.5. In liver mitochondria, the sensitivity to exogenous linolenate was considerably increased under such conditions. 4 nmol linolenate/mg protein proved enough to produce an almost complete release of the accumulated CaZi in KCl medium (as compared to 20 nmol in Fig. 3 ) . This increased sensitivity to linolenate could be related to the increased Ca2+load rather than
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Mitochondria
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Dependence of Cu2+Efflux on Linolencite Concmtrution
Fig. 4. t#kcts of Nu+ ions on ClPIi~C(CN)2-it~cluc.rd Cu2' releu.ve ,/;:om kidney mitochondricr. Incubation conditions as in Fig. 1 . (-- --) KCI medium; (- --) NaCl medium. 0.5 nmol C I P ~ Z C ( C N ) ~ / mg protein added at arrow. Incubation temperature 25 "C
Fig.6 shows the dependence of the Ca2+ efflux from liver mitochondria on the concentration of linolenate. The mitochondria were preloaded with Ca2+ (0.5 pmol/mg protein) in the presence of ATP and Pi, then linolenate was added and the Ca2+ efflux was measured 3 min later. In KCI medium, the Ca2+ release was complete at about 5 nmol linolenate/mg protein. In NaCl medium, 20- 25 nmol linolenate/mg protein were necessary to produce the complete Ca2+ release. Thus, the inhibition of the Ca2+ efflux by Na+ could be overcome by an increased linolenate concentration. Probably, at higher linolenate concentrations, its uncoupling activity becomes prevalent, the membrane potential collapses and the Ca2 efflux +
Kidney mitochondria
Liver mitochondria Linolenate 4 p M
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Fig. 5. EfJ2cls qflinolettic cic,id on the CuZ release /,om liver i A i cmd kit1ne.v i 6)mitochondricr under 'mcrssive lorrdirtg' conditiotls. Incubation medium contained: 120 m M KC1 or NaCI, 4 mM MgC12, 5 mM Hepes-Tris pH 7.2, 3 mM ATP-Na, 4 m M Pi, 3 mM Tris-succinate, 1 pM rotenone and 1 mg mitochondria1 protein/ml. Linolenic acid additions: liver mitochondria -4 nmoljmg protein. kidney miioKCI medium; (- -- -) NaCl medium. Incubation temperature 25 "C chondria - 20 nmol/mg protein. (--) +
I . Roman. P. Gmaj, C. Nowicka, and S. Angielski
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Fig. 6. C a z +release from liver mitochondria us ufutiction of linolenic acid concentration and ionic composition of the medium. Incubation conditions as in Fig. 5. ( 0 4 ) KCI medium; (A- ---A) NaCl medium. C a 2 + efflux was measured 3 min after the addition of linolenic acid. Incubation temperature 25 "C
Fig.8. Eflccts of saturated fatty acids on the c'o" release ,from liver mitochondriu. Incubation conditions as in Fig. 1. (. . . .) Lauric ) nmoll acid (C12),20 nmol/mg protein; (- ---) myristic acid ( C I ~20 mg protein; (- . -) palmitic acid (C16) 80 nmol/mg protein: (- -) hexanoic acid (C6) 80 nmol/mg protein. Incubation temperature 25 'C
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Fig.1. EJjccts o/ various unsaturated Jutty ucids on the Cuz+ release .from liver mitochondria. Incubation conditions as in Fig. 1. (. . . .) Arachidonic acid ( C Z O : ~ (-) ; - ) linolenic acid ( C I X : ~ )(-; . -) linoleic acid (C18:~);(--)oleic acid ( C I ~ : , )All . fatty acids added in a concentration of 24 nmol/mg protein
can no longer be inhibited by Na'. Apparently, the specific effect of linolenate on the Ca2+ transport can be observed at limited concentrations only. EJfects of DiJferent Fatty Acids on Ca2 Efflux In Fig. 7 and 8, the effects of a series of unsaturated and saturated fatty acids on the Ca2+ efflux from liver mitochondria are compared. All tested unsaturated fatty acids produced the Ca2+ efflux, in the following order of the effectiveness: arachidonic (Czo:4) > linolenic (C18:3) > linoleic (C18:2) > oleic ( C I ~ : I )In . all cases the fatty-acid-induced Ca2 efflux was inhibited by N a + ions (not shown). Both the long-chain palmitic acid (c16) and the short-chain hexanoic acid ((26) had +
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Fig, 9. EjJects of monovalent cations on the Ca2 rrlcase fiom l i v ~ r mitochondria. Incubation conditions as in Fig. 5 , except that KCI CsCI, . . . . NaCI. was replaced with equal concentrations of: KCl. Incubation temperature 25"C --- LiCI, +
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no effect on the Ca2+ efflux in concentrations up to 100 nmol/mg protein. On the other hand, the sturated fatty acids of a medium chain length, lauric (C12)and myristic (C,,) behaved similarly to the unsaturated fatty acids : they induced a Ca2+ efflux which had been inhibited by Na' ions. These results suggest that the length of the aliphatic chain of a fatty acid may be an essential factor in its effectiveness as a Ca2+-releasing compound. Unsaturated fatty acids may fold at cis double bonds and thus attain a chain length prerequisite for effectiveness. Effects of Monovalent Cations In Fig.9, the effects of the monovalent cations Li+, Na', K + and Cs' on the linolenate-induced Ca2+
Fatty-Acid-Induced CaZ Efflux from Mitochondria
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Fig. 10. Ejfects of' ruthenium red on the linolenate-induced Caz+ ejjlus from kidney mitochondria. Incubation conditions as in Fig. 1. (a) 1 pM ruthenium red added before mitochondria; (b) 1 pM ruthenium red added at 5 min (RR), 10 nmol linolenic acid/mg protein added at 6 m i n (LA); (c) 1 pM ruthenium red added at 5 min, no further additions
efflux from liver mitochondria are shown. The fastet Ca2+ efflux was observed in CsCl medium, a little slower one in KCI medium. In NaCl and LiCl media the Ca2+ efflux was inhibited. These results suggest that the radius (either hydrated or dehydrated) of a monovalent cation may determine its effects on Ca2+ transport. Effkcts of' Ruthenium Red on Linolenate-Induced Ca2' Efflux Ruthenium red is a well known non-competitive inhibitor of the energy-dependent Ca2 uptake by mitochondria, which acts by blocking the Ca2'binding glycoprotein involved in the electrophoretic Ca2+ uptake [7-91. The effects of linolenate on the Ca2+ efflux were thus measured in the presence of ruthenium red, since this might provide further evidence for the existence of independent routes of the Ca2+ uptake and release. The results are shown in Fig. 10. The concentrations of ruthenium red, sufficient to block the energy-dependent Ca2 uptake, failed to inhibit the linolenate-induced Ca2 efflux. This result strongly suggests that the linolenateinduced Ca2+ efflux does not proceed by simple reversal of the uptake pathway.
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Fig. 11. EfJects oflinolenic acid on the pussive siwllrng oJ'liver mitochondria inpotassium acetate andsodium acetate. Incubation medium contained 120 mM potassium acetate or sodium acetate, 1 pg/ml antimycin A and 1 mg/ml mitochondrial protein. lncubation temperature 25 'C. (- -)Control; (----) + linolenic acid 20 nmolimg protein
Fig. 12. E/fecrs of linolenic ucid and Cu2+on rlie etzergizod .swllitig mitochondria Incubation medium contained: 120 m M KCI (in A) or NaCI, CsCl or LiCl (in B), 5 mM Hepes-Tris, 0.5 mM MgC12, 3 m M Tris-succinate and 1 pM rotenone. Additions: 0.1 mM CaCI2, 24 nmol linolenic acidimg protein, 1 pM ruthenium red, 0.2 pM ClPhzC(CN)z. Incubation temperature 25 C uf liver
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Mitochondria1 Swelling during Linolenate-Induced Ca2' Release It was shown previously that unsaturated fatty acids and saturated fatty acids of medium chain length may induce an increased permeability of the inner mitochondrial membrane to monovalent cations, albeit at rather high concentrations [24]. Fig. 11 shows that linolenate in a concentration sufficient to produce
the Ca2+ release had no effect on the passive swelling of liver mitochondria in potassium acetate or sodium acetate, although at higher concentrations (e.g. 50 nmol/mg protein) the swelling was indeed observed. Thus, an increased permeability of the inner mitochondrial membrane to potassium can be excluded as the cause of the linolenate-induced Ca2' efflux. In Fig.12 the joined effects of linolenate and Ca2 on the energized swelling of liver mitochondria are shown. Neither linolenate alone nor Ca2+ alone produced mitochondrial swelling. In the presence of both linolenate and Ca2+ the mitochonria swelled extensively. The swelling induced by linolenate plus Ca2+ was inhibited by ruthenium red and by the uncoupler ClPhzC(CN)2 (Fig. 12A). Extensive swelling was observed in KCI and CsCl media only. In NaCl and LiCl media, i.e. under conditions when the Ca2+ efflux was inhibited, the swelling was inhibited as well. These data indicate that only a concerted action of Ca2+ and unsaturated fatty acid(s) produce mitochondrial swelling. Ca2+ must be recirculated across the inner mitochondrial membrane for the swelling +
I . Roman, P. Gmaj, C. Nowicka, and S. Angielski
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to occur, since it is inhibited both by the Ca2+uptake inhibitor ruthenium red and by the Ca2+ efflux inhibitors N a + and Li'. In addition, cyclic proton fluxes coupled to cyclic Ca2+ fluxes are probably involved, since the development of an independent route for the proton transport by means of added ClPhzC(CN)2 inhibits the swelling.
DISCUSSION The data presented here provide evidence that the unsaturated fatty acids and the saturated fatty acids of medium chain length (C12 and C14) can induce the Ca2+ efflux from kidney and liver mitochondria by a mechanism at least partly independent from their uncoupling activity. This is indicated by the following observations : (a) the fatty-acid-induced Ca2+ efflux is inhibited by Na+ and Li' ions, (b) the fatty-acidinduced uncoupling, as measured by the stimulation of oxygen uptake, is not influenced by N a + , and (c) the Ca2+ efflux induced by a classical uncoupler C I P ~ Z C ( C N )is~ insensitive to N a + . These results are not easily explained by the fatty-acid-induced uncoupling alone. It seems likely that in the presence of small concentrations of fatty acids the uncoupling is not complete, the increased respiration provides enough energy to fully compensate for an increased proton influx, and still the mitochondria maintain a negative potential of sufficient magnitude to drive Ca2+ in. Under such conditions, the mitochondria are able to reduce the medium Ca2+ concentration to normal low levels if only the Ca2' efflux has been inhibited by Na+ ions (Fig. 4). The specific inhibition by Na' of the fatty-acid-induced Ca2+ efflux can be observed only when relatively small concentrations of fatty acids have been used (Fig. 6). When higher concentrations of Unsaturated fatty acids have been added, the uncoupling prevailed and the Ca2+ efflux could no longer be inhibited by Na'. Possibly, the Ca2+ efflux from uncoupled mitochondria proceeds via an Na+-insensitive reversal of the uptake pathway. The same might be true for the anaerobic Ca2 efflux (Fig. 3). A transition to anaerobiosis with the consequent disappearance of the membrane potential resulted in a prompt and complete release of all the accumulated Ca2' in both KCI and NaCl media. N o consistent effects of N a + on the anaerobic Ca2+ efflux could be observed, which suggested that it might proceed via a reversal of the uptake pathway. The Ca2+ uptake into and its release from the mitochondria could be distinguished by means of the selective inhibition : ruthenium red inhibited completely the Ca2+ uptake but had no effect on the fatty-acid-induced Ca2+ release (Fig. 10); N a + and Li+ ions inhibited both the spontaneous Ca2' efflux +
Fig. 13. Proposed mechanism of' uction of unsuiururrrl f u r r ~acids on the Cuz+ efflux and mitochondria1 .nve/Iing. For explanation see text
from kidney mitochondria and the fatty-acidinduced Ca2+ efflux from liver mitochondria, but had no effect on the Ca2+ uptake. These data may be interpreted as indicating that the Ca2+uptake and the Ca2+ release proceed via independent pathways, the latter being activated or catalyzed by endogenous or exogenous Unsaturated fatty acids. Some clues to the mechanism of the fatty-acidinduced Ca2+ efflux were provided by the swelling experiments. It was shown (Fig. 12) that linolenate in the presence of Ca2+ and of the energy source (succinate) induced a mitochondrial swelling which was inhibited by both the Ca2+ uptake inhibitor ruthenium red and the Ca2+efflux inhibitors Na' and Li+. These data indicated that the cyclic Ca2+ flux across the mitochondrial membrane was the primary event in the induction of mitochondrial swelling. In addition, the inhibition of swelling by a proton conductor CIPhzC(CN)2 suggested the involvement of proton fluxes in the mechanism of swelling. These observations led us to propose a tentative mechanism of action of unsaturated fatty acids which is illustrated in Fig.13. The unsaturated fatty acids and the saturated fatty acids of an appropriate chain length can dissolve in the membrane core and traverse it in the undissociated form. Ca2+ is taken up by the ruthenium-red-sensitive electrophoretic mechanism and is accumulated in the matrix space (Fig. 13A). On the inner surface of the membrane, Ca2+ binds with carboxyl groups of two molecules of fatty acids with the concomitant release of two H + ions into the matrix space-a reaction facilitated by high Ca2+ and low H + concentrations in the matrix (Fig. 13B). The Ca2+/fatty acid complex diffuses in the direction of a low Ca2+ concentration in the outer space (Fig. 13C). The accumulation of H + in the matrix induces a coupled H + / K + exchange [25]. The operation of such a cycle results in the net accumulation of K + ions in the matrix space. The entry of an anion (e.g. succinate) completes the conditions required for the osmotic swelling. The proposed mechanism is, in essence, an electroneutral Ca2+/H' exchange in which unsaturated fatty
Fatty-Acid-Induced Ca2+ Efflux from Mitochondria
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acids (or medium-chain-length saturated fatty acids) act as mobile Ca2+ and H + carriers. The electroneutral C a 2 + / H + exchange was already postulated by other authors as a mechanism of the Ca2+ efflux from liver mitochondria [18,22]. The inhibition by Na+ of the Ca2+ efflux can conveniently by explained by a competition between the two ions for binding with fatty acids at the inner surface of the membrane which is readily permeable to Na' [23] (Fig.l2), although other mechanisms cannot be excluded at present. An alternative possibility is that the unsaturated fatty acids increase the fluidity of the mitochondrial membrane and thereby activate an endogenous Ca2 carrier which catalyzes the CaZ efflux. Indeed, we have observed that the spontaneous Ca2 efflux from kidney mitochondria was stimulated by the agents which increased the membrane fluidity (e.g. diethyl ether), and it was inhibited by the agents which decreased the membrane fluidity (e.g. MgC12) (data not shown). Also, the spontaneous Ca2+ efflux was strongly activated when the incubation temperature was raised above 23 -25 However, the interpretation of such results is difficult since most factors which increase the membrane fluidity, stimulate the mitochondrial phospholipase A as well [26]. Therefore, on the basis of Ca2+ transport measurements alone it cannot be decided whether the increased Ca2+efflux is due to the activation of endogenous ionophores or due to increased fatty acid concentration in the mitochondrial membranes resulting from phospholipase A2 stimulation. In addition, if fatty acids acted as mobile Ca2+ carriers, their effects on the Ca2+ transport would be expected to depend on the membrane fluidity. It may be noted, however, that the powerful inhibition of the spontaneous Ca2+ efflux from kidney mitochondria by albumin, even at high incubation temperatures, strongly suggest that the endogenous Ca2+ ionophores might be fatty acids or their derivatives (see also [ZS]). The observation that N a + ions inhibit the spontaneous Ca2 efflux from freshly prepared kidney mitochondria (Fig. 2) suggests that the endogenous fatty acids may indeed catalyse the Ca2+ efflux in vivo. It is the membrane phospholipids which are the most likely source of the mitochondrial Unsaturated fatty acids. Most phospholipids contain unsaturated fatty acids in position C2 of glycerol, and the mitochondria show considerable activity of phospholipase A2 which hydrolyses the phospholipids in C2 position [26,27]. The further fates of the split fatty acids, however, and the control of their level in the mitochondrial membranes, make as yet an almost completely obscure picture. The inhibition by sodium of the Ca2+ efflux from kidney mitochondria may have some physiological significance. In kidney cells the extrusion of Ca2+from +
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the cell to the interstitial fluid proceeds via an Na'/ Ca2+ exchange mechanism: the Ca2+efflux from the cell is coupled to the Na+ influx into the cell [29]. The inhibition by Na+ of mitochondrial Ca2+efflux would provide a negative feedback loop between the Ca2 transport in the plasma membrane and in mitochondria. This is just the opposite to the positive feedback observed in the heart [15,16]. Such a mechanism is not unreasonable, however, if we realize that the kidney cells do transport large amounts of Ca2+ from the tubule lumen to the peritubular fluid. In this situation, the inhibition by sodium of mitochondrial Ca2+ efflux may be essential for keeping the cytosolic Ca2+ at low levels in face of varying loads of Ca2+ transported across the cell. +
This work was supported in part by grant 11. 1.1.5 from the Polish Academy of Sciences.
REFERENCES 1. Rasmussen, H., Goodman, D. B. P., Friedman, N., Allen, J . E. & Kurokawa, K. (1976) in Handbook of Physiology, Endocrinology (Creep, R . 0. & Astwood, E. B., eds) vol. 7. pp. 225 - 263, American Physiological Society, Washington D.C. 2. Borle, A. B. (1973) Fed. Proc. 32, 1944-1950. 3. Lehninger, A. L., Carafoli, E. & Rossi, C. S. (1967) Adv. Enzymol. 29, 259 - 320. 4. Carafoli, E. & Lehninger, A. L. (1971) Biochem. J . 122, 681 690. 5. Rottenberg, H . B Scarpa, A. (1974) Biochemistry, 13, 481 1 4817. 6. Heaton, G. M . & Nicholls, D. J. (1976) Biochem. J . 156, 639646. 7. Sottocasa, G., Sandri, G., Panfili, E., de Bernard, B., Grazzotti, P., Vasington, F. D. & Carafoli, E. (1972) Biochem. Biophys. Res. Commun. 47, 808-813. 8. Mela, L. (1968) Arch. Biochem. Biophys. 123, 286-291. 9. Vasington, F. D., Grazzotti, P., Tiozzo, R. & Carafoli, E. (1972) Biochim. Biophys. Aciu, 256, 43 - 54. 10. Perstipino, G . , Panfili, E., Conti, F. & Carafoli, E. (1974) FEBS Lett. 45, 99- 103. 11. Azarashwili, T. S., Lukyanienko, A. I. & Evtodicnko, Yu. V. (1978) Biokhimia, 43, 1139-1142. 12. Pozzan, T., Bragadin, M. & Azzone, G . F. (1977) Biochemistry, 16,5618-5625. 13. Harris, E. J. (1977) Biochem. J . 168, 447-456. 14. Caroni, P., Schwerzmann, K. & Carafoli, E. (1978) FEBS Leit. 96,339- 342. 15. Crompton, M., Capano, M. & Carafoli, E. (1977) Eur. J . Biochem. 69, 453 - 462. 16. Crompton, M., Kunzi, M . & Carafoli, E. (1977) Eur. J . Biochem. 79, 549- 558. 17. Crompton, M., Moser, R. L., Ludi, M. Sr Carafoli, E. (1978) Eur. J . Biochem. 82, 25 - 31, 18. Drdhota, Z., Carafoli, E., Rossi, C. S., Gamble, R. L. & Lehninger, A. L. (1965) J . B i d . C'hem. 240, 2712-2720. 19. Lehninger, A. L., Vercesi, A. & Bababunmi, E. A. (1978) Proc. Nail Acad. Sci. U.S.A. 75, 1690-1694. 20. Pressman, B. C. & Lardy, H. A. (1955) Biochem. Biophys. Actu, 18,482- 487. 21. Borst, P., Loos, J. A,, Christ, E. J. & Slater, E. G . (1962) Biochim. Biophys. Acta, 62, 509-518.
I. Roman, P. Gmaj, C. Nowicka, and S. Angielski 22. Puskin, J. S., Gunter, T. E., Gunter, K. K. & Russel, P. R. (1 976) Biochemistry, 15, 3834- 3842. 23. Douglas, M. G. & Cockrell, R. S. (1974) J . Biol. Chem. 249, 5462 - 5471. 24. Wojtczak. L. (1974) FEBS Lett. 44, 25-30. 25. Massari, S. Cuc Azzone, G. F. (1970) Eur. J . Biochem. 12, 301309.
623 26. Vignais, P. M., Nachbaur, J. & Colbeau, A. (1971) in Mrmhvane Bound Enzym~5 (Porcellati, G. & doJoss, F., eds) pp. 87- 109, Plenum Press, New York. 27. Scherphof, G. L. & van Deenen, L. L. M. (1965) Biochim. Biophys. Acta, Y8, 204-206. 28. Blondin, G. A. (1975) Ann. N . Y . Acad. Sci. 164, 98-111. 29. Gmaj, P., Murer, H. & Kinne, R. (1979) Biochern. J . 178. 549 - 557.
I. Roman, Division of Neurology, RG-20, University of Washington, School of Medicine, Seattle, Washington, U.S.A. 98195 P. Gmaj*, C. Nowicka, and S. Angielski, Zaklad Biochemii Klinicznej, Instytut Patologii, Akademia Medyczna, PL-80-211 Gdansk, Poland
* To whom corrcspondence
should be addressed
Regulation of Ca2+Efflux from Kidney and Liver Mitochondria by Unsaturated Fatty Acids and Na' Ions Izabela ROMAN, Piotr GMAJ, Cecylia NOWICKA, and Stefan ANGIELSKI Department of Clinical Biochemistry, Institute of Pathology, Medical Academy, Gdai7sk (Received January 30/August 28, 1979)
The effects of fatty acids and monovalent cations on the Ca2+efflux from isolated liver and kidney mitochondria were investigated by means of electrode techniques. It was shown that unsaturated fatty acids and saturated fatty acids of medium chain length (CIZ and C I ~ induced ) a Ca2+ efflux from mitochondria which was not inhibited by ruthenium red, but was specifically inhibited by Na' and Li'. The Ca2+-releasingactivity of unsaturated fatty acids did not correlate with their uncoupling activity. In kidney mitochondria a spontaneous, temperature-dependent Ca2 efflux was observed which was inhibited either by albumin or by Na'. It is suggested that the net Ca2+ accumulation by mitochondria depends on the operation of independent pump and leak pathways. The pump is driven by the membrane potential and can be inhibited by ruthenium red, the leak depends on the presence of unsaturated fatty acids and is inhibited by Na' and Li'. It is suggested that the unsaturated fatty acids produced by mitochondrial phospholipase A2 can be essential in the regulation of the Ca2+ retention in and the Ca2+ release from the mitochondria. +
The role of mitochondria in the maintenance of the steady state Ca2' concentration in kidney cells is well documented [1,2]. However, of the two processes involved in net calcium transport, namely the Ca2+ uptake and the Ca2+ release, only the former had been subjected to thorough investigations [3,4]. It is now generally accepted that the driving force for the mitochondrial Ca2+ uptake is provided by the electric field generated across the inner mitochondrjal membrane by respiration or ATP hydrolysis [5,6], and that the process is catalysed by a specific Ca2+-binding glycoprotein located in the inner mitochondrial membrane and inhibited by ruthenium red and lanthanides [7-91. The Ca2+ transport was reconstituted by the incorporation of the isolated mitochondrial Ca2+-binding glycoprotein into artificial lipid bilayer membranes, whereby its role in Ca2+ transport was confirmed [lo, 111. The stoichiometry of charge transfer accompanying the Ca2 uptake was a matter of some debate. Thus, direct measurements under conditions of limited Ca2+ loading provided the n value of 2 [ 5 , 6 ] ,but deviations from the Nernstian Ca2' distribution were commonly encountered in measurements of the Ca2+ accumulation [12]. A problem of independent pathways for +
Abbreviations. ClPhzC(CN)*, carbonylcyanide m-chlorophenylhydrazone; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; EGTA, [ethylenebis(oxoethylenenitrilo)]tetraaceticacid.
the Ca2+ uptake and release had thus been put forward [13,14]. In the mitochondria from heart and from other excitable tissues a specific Na"/Ca2 antiporter was identified which might operate as a Ca2+-releasing mechanism [15-171. This antiporter is absent in the mitochondria from kidney and liver, and thus the pathways of the Ca2+ efflux from these mitochondria remain to be identified. The retention of accumulated Ca2+ in mitochondria provides a closely related problem. Normally, in the absence of ATP, Ca2+ is retained in the isolated mitochondria for a brief period of time only, and subsequently it is released into the suspending medium in an auto-accelerating process [13,18]. The Ca2+ retention and release depends on several seemingly unrelated external factors such as the Ca2+ to protein ratio, the ionic composition of the medium, the presence or absence of phosphate, the oxidation/ reduction of pyridine nucleotides, and others [12,18, 191. The basic mechanisms which control the Ca2+ retention, however, are unknown. In this paper the data are presented which indicate that the endogenous or exogenous unsaturated fatty acids and saturated fatty acids of medium chain length may be involved in the catalysis of CaZ+ 'leak' from mitochondria by a mechanism partly independent of their uncoupling activity. The Ca2+ efflux induced by unsaturated fatty acids is specifically inhibited +
Fatty-Acid-Induced C a 2 + Efflux from Mitochondria
616
by Na', what may provide an important regulatory link in the intracellular Ca2+ homeostasis.
Mitochondria
MATERIALS AND METHODS Mitochondria were isolated from the liver and from the kidney cortex of female Wistar rats by a conventional differential centrifugation procedure. The tissue was disrupted in a Potter homogenizer with a teflon pestle in a medium containing 0.25 M sucrose, 0.01 M Tris-C1 buffer pH 7.4 and 0.1 mM EGTA. The mitochondrial pellets were washed three times. In the last two washes no EGTA was used. The mitochondrial protein was measured by a biuret method with bovine serum albumin as standard. The standard incubation medium in a final volume of 2.5 ml contained: 120 mM KCI or NaCl (see legends), 5 mM Hepes-Tris buffer pH 7.2, 0.5 mM MgC12, 3 mM Tris-succinate, 1 pM rotenone and 1 mg mitochondrial protein per ml. CaC12 was added for either 0.1 mM or 0.5 mM final concentration (limited loading and massive loading conditions, respectively). Fatty acids were added as freshly prepared ethanolic solutions, kept at 0 ° C until used. Other additions were (see legends for the figures): bovine serum albumin (essentially fdtty-acid-free, Sigma Chem. Co., St Louis, Mo., U.S.A.) 5 mg/ml; Na-ATP 3 mM; ruthenium red (Sigma) 1 p M ; CIPhzC(CN)2 (Calbiochem) 0.5 pM. Medium Ca2+ was continuously monitored with a Ca2+-sensitive electrode (F-2112, Radiometer, Denmark) connected to a pH-meter, antilogarithmic amplifier and recorder. H movements and oxygen uptake were measured simultaneously with a glass and a Clark-type oxygen electrodes, respectively. All electrodes were mounted in the same closed, thermostatted reaction chamber. The responses of the Ca2+ and H electrodes were calibrated before each measurement. To simplify the drawings, H + and O2 traces are shown only when relevant information has been provided. Mitochondria1 swelling was measured by recording the changes in absorbance of mitochondrial suspensions at 520 nm in a Zeiss-Opton recording spectrophotometer. All reagents used were of analytical grade. +
+
RESULTS Fjfects of Temperature on Energy-Dependent Uptake, Retention and Release o j Cu2+by Kidney Mitochondria
In the energy-linked uptake, the Ca2+ distribution between the mitochondria and the suspending medium reaches a steady state which presumably is maintained by the equilibrium between the Ca2+ uptake
Fig. 1. Temperature dependence of rhe Ca" uptake and release by kidney mitochondria. Incubation medium contained: 120 m M KC1, 0.5 mM MgC12, 5 mM Hepes-Tris pH 7.2, 3 mM Tris-succinate. 1 pM rotenone and 100 pM CaC12. The Ca2+ uptake was started by addition of kidney mitochondria 1 mg/ml
and release. Fig. 1 shows that the steady state of Ca2+ distribution and the retention of Ca2+ by kidney mitochondria are powerfully influenced by temperature. At 20 "C practically all Ca2+ added (100 nmol/mg protein) was taken up and retained until oxygen had been exhausted, i.e. for at least 8 min. As the temperature was raised a progressively smaller proportion of the total Ca2+ present was taken up and, after a period of steady-state maintenance which was the shorter the higher the temperature had been, a spontaneous release of the accumulated Ca2+ was clearly seen. Sharp upper deflection points in Fig. 1 correspond to the exhaustion of oxygen in the suspending medium. Apparently, the inside negative membrane potential disappears during anaerobiosis, what results in a rapid and complete release of the accumulated Ca2+.The initial rate of the Ca2+ uptake, on the other hand, was much less dependent on temperature than the Ca2+ release. This might suggest that the Ca2+ uptake and efflux are catalysed by systems with different temperature dependence. The temperaturedependent Ca2+ release from liver mitochondria, albeit observable, was much less pronounced than that from kidney mitochondria. This discrepancy will be discussed later on. Eflects o f Sodium and Bovine Serum Albumin
Fig.2 shows that the usual, spontaneous Ca" release from kidney mitochondria incubated at 30 "C can be abolished if K + is substituted with N a + as the main cation in the medium. In NaCl medium, the amount of Ca2+ taken up was considerably greater, the period of the steady-state retention having been prolonged, and the efflux phase much less pronounced. When bovine serum albumin (essentially fatty-acidfree) was added to the medium, the spontaneous CaZ+ release disappeared completely : the mitochondria took up all the Ca2+ added and retained it until oxygen had been exhausted. In the presence of bovine
I. Roman, P. Gmaj, C. Nowicka, and S. Angielski
617
Mitochondria
Liver mitochondria
4
0'
Kidnev mitochondria
lmin
Fig.2. Eflects of' Na' ions and albumin on the Ca2' uptake and releasc by kidney mitochondria. Experimental conditions as in Fig. I . ( ~--) KCI medium; (----) 120 m M NaCl was substituted for KCI in the medium. Albumin concentration 5 mg/ml. Incubation temperature 30 C
0
2
4
6
Time (min)
serum albumin, Na+ ions were of no effect on both Ca2 uptake and release. These experiments allowed for the following conclusions: (a) the spontaneous temperature-dependent Ca2 release from kidney mitochondria depends on the presence of some factor removable by bovine serum albumin, probably fatty acids, and (b) N a + ions inhibit the fatty-acid-dependent Ca2+ efflux, but have no effect on the Ca2+ uptake, having been ineffective in the presence of bovine serum albumin. Therefore, in further experiments the effects of exogenous fatty acids and N a + ions on the Ca2+ release were investigated in more detail. +
+
E f f k t s of' Exogenous Linolenic Acid and Sodium Ions on Cu2+Elflux born Kidney and Liver Mitochondriu In Fig. 3 the effects of exogenous linolenic acid on the Ca2+ release from liver and kidney mitochondria are shown. The experiments were performed at 25 "C to avoid a large spontaneous Ca2 release. Linolenate as added after the Ca2+ uptake had been completed, evoked an immediate, graded Ca2+ release from both liver (Fig. 3A) and kidney (Fig. 3B) mitochondria. A few minutes after linolenate had been added, a new steady state was established at a much lower Ca2+ accumulation level. Upon exhaustion of oxygen from the medium the residual mitochondria1 Ca2 was immediately and completely released. In both liver and kidney mitochondria the Ca2 efflux induced by linolenate was inhibited by Na+ ions. Thus, the addition of linolenate at 25 "C closely mimicked the spontaneous Ca2 release observed in kidney mitochondria at higher temperatures (compare Fig.2 and 3). In +
+
+
+
8
'
0
2
4
6
8
Time (min)
Fig. 3. Efiects of' linolenic acid (C18 3 ) arid No+ ions on rhr Ca2 release .from liver ( A ) and kidney ( B ) mitochondria. Incubation KCI medium, (-----) NaCl medium. conditions as in Fig. 1. (-) Linolenic acid added as ethanolic solutions in concentrations: liver mitochondria -20 nmol/mg protein, kidney mitochondria - 10 nmol/mg protein. Incubation temperature 25 C +
liver, as compared to kidney mitochondria, the concentration of linolenate necessary to evoke Ca2 release was approximately twice as high. A lower sensitivity to exogenous linolenate and a lower spontaneous Ca2 release suggest that the endogenous fatty acid level in liver mitochondria is low. (Direct measurements have shown that the levels of endogenous free fatty acids are 3 to 10 times higher in kidney than in liver mitochondria, depending on the fatty acid species: unpublished results of P. Gmaj.) In kidney mitochondria, on the other hand, the effects of endogenous and exogenous fatty acids may sum up producing a higher rate of the Ca2+ efflux. The bottom panels of F i g 3 show that the mitochondrial respiration is activated upon an addition of linolenate. This is the well known uncoupling activity of unsaturated fatty acid [20,21]. However, the stimulation of respiration is identical in both NaCl and KCI media. Thus, the Ca2+ release induced by linolenate cannot be attributed solely to its uncoupling activity and to the collapse of the membrane potential: an inhibition of the Ca2+ efflux by N a + indicates a specific effect of linolenate on the Ca2+ transport. This conclusion had been substantiated by the experiment shown in Fig.4. The Ca2+ efflux from kidney mitochondria induced by a classical uncoupler CIPhzC(CN)2 was not influenced by N a +
+
+
Fatty-Acid-Induced Ca2+ Efflux from Mitochondria
618
to the presence of ATP or Pi, since it was drastically decreased upon lowering of the Ca2+ to 0.1 pmol/mg protein (not shown). As in previous experiments, sodium ions strongly inhibited the linolenate-induced Ca2+efflux. In kidney mitochondria, on the contrary, the sensitivity to exogenous linolenate was decreased in the presence of ATP, and higher linolenate concentrations were necessary to induce Ca2+ release than those under limited loading conditions. The interpretation of these results are not quite clear yet, but they suggest that the rate of the linolenate-induced Ca2+ efflux from liver mitochondria depends on the amount of Ca2 accumulated in the mitochondrial matrix: when it is increased, the Ca2+ efflux rate at a given level of linolenate becomes increased, too. In kidney mitochondria, the added linolenate may possibly be metabolished in ATP-dependent reaction(s). This organ specificity requires further investigations.
ions. In the latter case, the CaL+release might indeed result directly from the collapse of the membrane potential. Efects o j Linolenute in the Presence o f A T P and Pi The effects of linolenate on the Ca2+ efflux from kidney and liver mitochondria were further investigated under 'massive loading' conditions (0.5 pmol Ca2+/mg protein) in the presence of ATP and inorganic phosphate. The results are shown in Fig.5. In liver mitochondria, the sensitivity to exogenous linolenate was considerably increased under such conditions. 4 nmol linolenate/mg protein proved enough to produce an almost complete release of the accumulated CaZi in KCl medium (as compared to 20 nmol in Fig. 3 ) . This increased sensitivity to linolenate could be related to the increased Ca2+load rather than
+
Mitochondria
I
Dependence of Cu2+Efflux on Linolencite Concmtrution
Fig. 4. t#kcts of Nu+ ions on ClPIi~C(CN)2-it~cluc.rd Cu2' releu.ve ,/;:om kidney mitochondricr. Incubation conditions as in Fig. 1 . (-- --) KCI medium; (- --) NaCl medium. 0.5 nmol C I P ~ Z C ( C N ) ~ / mg protein added at arrow. Incubation temperature 25 "C
Fig.6 shows the dependence of the Ca2+ efflux from liver mitochondria on the concentration of linolenate. The mitochondria were preloaded with Ca2+ (0.5 pmol/mg protein) in the presence of ATP and Pi, then linolenate was added and the Ca2+ efflux was measured 3 min later. In KCI medium, the Ca2+ release was complete at about 5 nmol linolenate/mg protein. In NaCl medium, 20- 25 nmol linolenate/mg protein were necessary to produce the complete Ca2+ release. Thus, the inhibition of the Ca2+ efflux by Na+ could be overcome by an increased linolenate concentration. Probably, at higher linolenate concentrations, its uncoupling activity becomes prevalent, the membrane potential collapses and the Ca2 efflux +
Kidney mitochondria
Liver mitochondria Linolenate 4 p M
I
500
I
I
Linolenate
20pM
I
- 400 5 =L
I
A
2 .-5
300
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0 2
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4 6 Time (min)
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10
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4 6 Time (min)
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Fig. 5. EfJ2cls qflinolettic cic,id on the CuZ release /,om liver i A i cmd kit1ne.v i 6)mitochondricr under 'mcrssive lorrdirtg' conditiotls. Incubation medium contained: 120 m M KC1 or NaCI, 4 mM MgC12, 5 mM Hepes-Tris pH 7.2, 3 mM ATP-Na, 4 m M Pi, 3 mM Tris-succinate, 1 pM rotenone and 1 mg mitochondria1 protein/ml. Linolenic acid additions: liver mitochondria -4 nmoljmg protein. kidney miioKCI medium; (- -- -) NaCl medium. Incubation temperature 25 "C chondria - 20 nmol/mg protein. (--) +
I . Roman. P. Gmaj, C. Nowicka, and S. Angielski
619 Mitochondria
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7
Fatty acid
I
\; ///-
---____---
I
,
1
0
2
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8
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12
Time (rnin)
Fig. 6. C a z +release from liver mitochondria us ufutiction of linolenic acid concentration and ionic composition of the medium. Incubation conditions as in Fig. 5. ( 0 4 ) KCI medium; (A- ---A) NaCl medium. C a 2 + efflux was measured 3 min after the addition of linolenic acid. Incubation temperature 25 "C
Fig.8. Eflccts of saturated fatty acids on the c'o" release ,from liver mitochondriu. Incubation conditions as in Fig. 1. (. . . .) Lauric ) nmoll acid (C12),20 nmol/mg protein; (- ---) myristic acid ( C I ~20 mg protein; (- . -) palmitic acid (C16) 80 nmol/mg protein: (- -) hexanoic acid (C6) 80 nmol/mg protein. Incubation temperature 25 'C
Mitochondria
V
Mitochondria
I 1
7
Fatty acid
V
_..._...' /
,
/
0
i
4
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16
1 2 1 4
Time (rnin)
Fig.1. EJjccts o/ various unsaturated Jutty ucids on the Cuz+ release .from liver mitochondria. Incubation conditions as in Fig. 1. (. . . .) Arachidonic acid ( C Z O : ~ (-) ; - ) linolenic acid ( C I X : ~ )(-; . -) linoleic acid (C18:~);(--)oleic acid ( C I ~ : , )All . fatty acids added in a concentration of 24 nmol/mg protein
can no longer be inhibited by Na'. Apparently, the specific effect of linolenate on the Ca2+ transport can be observed at limited concentrations only. EJfects of DiJferent Fatty Acids on Ca2 Efflux In Fig. 7 and 8, the effects of a series of unsaturated and saturated fatty acids on the Ca2+ efflux from liver mitochondria are compared. All tested unsaturated fatty acids produced the Ca2+ efflux, in the following order of the effectiveness: arachidonic (Czo:4) > linolenic (C18:3) > linoleic (C18:2) > oleic ( C I ~ : I )In . all cases the fatty-acid-induced Ca2 efflux was inhibited by N a + ions (not shown). Both the long-chain palmitic acid (c16) and the short-chain hexanoic acid ((26) had +
I
0
1
2 3 Time (min)
4
5
Fig, 9. EjJects of monovalent cations on the Ca2 rrlcase fiom l i v ~ r mitochondria. Incubation conditions as in Fig. 5 , except that KCI CsCI, . . . . NaCI. was replaced with equal concentrations of: KCl. Incubation temperature 25"C --- LiCI, +
~
no effect on the Ca2+ efflux in concentrations up to 100 nmol/mg protein. On the other hand, the sturated fatty acids of a medium chain length, lauric (C12)and myristic (C,,) behaved similarly to the unsaturated fatty acids : they induced a Ca2+ efflux which had been inhibited by Na' ions. These results suggest that the length of the aliphatic chain of a fatty acid may be an essential factor in its effectiveness as a Ca2+-releasing compound. Unsaturated fatty acids may fold at cis double bonds and thus attain a chain length prerequisite for effectiveness. Effects of Monovalent Cations In Fig.9, the effects of the monovalent cations Li+, Na', K + and Cs' on the linolenate-induced Ca2+
Fatty-Acid-Induced CaZ Efflux from Mitochondria
620
+
Mitochondria
Mitochondria
V
V
-------- -- - - 'acetate Potassium -----
--I
lrnin
I
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6 8 Time (mini
1 0 1 2
14
Fig. 10. Ejfects of' ruthenium red on the linolenate-induced Caz+ ejjlus from kidney mitochondria. Incubation conditions as in Fig. 1. (a) 1 pM ruthenium red added before mitochondria; (b) 1 pM ruthenium red added at 5 min (RR), 10 nmol linolenic acid/mg protein added at 6 m i n (LA); (c) 1 pM ruthenium red added at 5 min, no further additions
efflux from liver mitochondria are shown. The fastet Ca2+ efflux was observed in CsCl medium, a little slower one in KCI medium. In NaCl and LiCl media the Ca2+ efflux was inhibited. These results suggest that the radius (either hydrated or dehydrated) of a monovalent cation may determine its effects on Ca2+ transport. Effkcts of' Ruthenium Red on Linolenate-Induced Ca2' Efflux Ruthenium red is a well known non-competitive inhibitor of the energy-dependent Ca2 uptake by mitochondria, which acts by blocking the Ca2'binding glycoprotein involved in the electrophoretic Ca2+ uptake [7-91. The effects of linolenate on the Ca2+ efflux were thus measured in the presence of ruthenium red, since this might provide further evidence for the existence of independent routes of the Ca2+ uptake and release. The results are shown in Fig. 10. The concentrations of ruthenium red, sufficient to block the energy-dependent Ca2 uptake, failed to inhibit the linolenate-induced Ca2 efflux. This result strongly suggests that the linolenateinduced Ca2+ efflux does not proceed by simple reversal of the uptake pathway.
.'\%dim
acetate
Fig. 11. EfJects oflinolenic acid on the pussive siwllrng oJ'liver mitochondria inpotassium acetate andsodium acetate. Incubation medium contained 120 mM potassium acetate or sodium acetate, 1 pg/ml antimycin A and 1 mg/ml mitochondrial protein. lncubation temperature 25 'C. (- -)Control; (----) + linolenic acid 20 nmolimg protein
Fig. 12. E/fecrs of linolenic ucid and Cu2+on rlie etzergizod .swllitig mitochondria Incubation medium contained: 120 m M KCI (in A) or NaCI, CsCl or LiCl (in B), 5 mM Hepes-Tris, 0.5 mM MgC12, 3 m M Tris-succinate and 1 pM rotenone. Additions: 0.1 mM CaCI2, 24 nmol linolenic acidimg protein, 1 pM ruthenium red, 0.2 pM ClPhzC(CN)z. Incubation temperature 25 C uf liver
+
+
+
Mitochondria1 Swelling during Linolenate-Induced Ca2' Release It was shown previously that unsaturated fatty acids and saturated fatty acids of medium chain length may induce an increased permeability of the inner mitochondrial membrane to monovalent cations, albeit at rather high concentrations [24]. Fig. 11 shows that linolenate in a concentration sufficient to produce
the Ca2+ release had no effect on the passive swelling of liver mitochondria in potassium acetate or sodium acetate, although at higher concentrations (e.g. 50 nmol/mg protein) the swelling was indeed observed. Thus, an increased permeability of the inner mitochondrial membrane to potassium can be excluded as the cause of the linolenate-induced Ca2' efflux. In Fig.12 the joined effects of linolenate and Ca2 on the energized swelling of liver mitochondria are shown. Neither linolenate alone nor Ca2+ alone produced mitochondrial swelling. In the presence of both linolenate and Ca2+ the mitochonria swelled extensively. The swelling induced by linolenate plus Ca2+ was inhibited by ruthenium red and by the uncoupler ClPhzC(CN)2 (Fig. 12A). Extensive swelling was observed in KCI and CsCl media only. In NaCl and LiCl media, i.e. under conditions when the Ca2+ efflux was inhibited, the swelling was inhibited as well. These data indicate that only a concerted action of Ca2+ and unsaturated fatty acid(s) produce mitochondrial swelling. Ca2+ must be recirculated across the inner mitochondrial membrane for the swelling +
I . Roman, P. Gmaj, C. Nowicka, and S. Angielski
621
to occur, since it is inhibited both by the Ca2+uptake inhibitor ruthenium red and by the Ca2+ efflux inhibitors N a + and Li'. In addition, cyclic proton fluxes coupled to cyclic Ca2+ fluxes are probably involved, since the development of an independent route for the proton transport by means of added ClPhzC(CN)2 inhibits the swelling.
DISCUSSION The data presented here provide evidence that the unsaturated fatty acids and the saturated fatty acids of medium chain length (C12 and C14) can induce the Ca2+ efflux from kidney and liver mitochondria by a mechanism at least partly independent from their uncoupling activity. This is indicated by the following observations : (a) the fatty-acid-induced Ca2+ efflux is inhibited by Na+ and Li' ions, (b) the fatty-acidinduced uncoupling, as measured by the stimulation of oxygen uptake, is not influenced by N a + , and (c) the Ca2+ efflux induced by a classical uncoupler C I P ~ Z C ( C N )is~ insensitive to N a + . These results are not easily explained by the fatty-acid-induced uncoupling alone. It seems likely that in the presence of small concentrations of fatty acids the uncoupling is not complete, the increased respiration provides enough energy to fully compensate for an increased proton influx, and still the mitochondria maintain a negative potential of sufficient magnitude to drive Ca2+ in. Under such conditions, the mitochondria are able to reduce the medium Ca2+ concentration to normal low levels if only the Ca2' efflux has been inhibited by Na+ ions (Fig. 4). The specific inhibition by Na' of the fatty-acid-induced Ca2+ efflux can be observed only when relatively small concentrations of fatty acids have been used (Fig. 6). When higher concentrations of Unsaturated fatty acids have been added, the uncoupling prevailed and the Ca2+ efflux could no longer be inhibited by Na'. Possibly, the Ca2+ efflux from uncoupled mitochondria proceeds via an Na+-insensitive reversal of the uptake pathway. The same might be true for the anaerobic Ca2 efflux (Fig. 3). A transition to anaerobiosis with the consequent disappearance of the membrane potential resulted in a prompt and complete release of all the accumulated Ca2' in both KCI and NaCl media. N o consistent effects of N a + on the anaerobic Ca2+ efflux could be observed, which suggested that it might proceed via a reversal of the uptake pathway. The Ca2+ uptake into and its release from the mitochondria could be distinguished by means of the selective inhibition : ruthenium red inhibited completely the Ca2+ uptake but had no effect on the fatty-acid-induced Ca2+ release (Fig. 10); N a + and Li+ ions inhibited both the spontaneous Ca2' efflux +
Fig. 13. Proposed mechanism of' uction of unsuiururrrl f u r r ~acids on the Cuz+ efflux and mitochondria1 .nve/Iing. For explanation see text
from kidney mitochondria and the fatty-acidinduced Ca2+ efflux from liver mitochondria, but had no effect on the Ca2+ uptake. These data may be interpreted as indicating that the Ca2+uptake and the Ca2+ release proceed via independent pathways, the latter being activated or catalyzed by endogenous or exogenous Unsaturated fatty acids. Some clues to the mechanism of the fatty-acidinduced Ca2+ efflux were provided by the swelling experiments. It was shown (Fig. 12) that linolenate in the presence of Ca2+ and of the energy source (succinate) induced a mitochondrial swelling which was inhibited by both the Ca2+ uptake inhibitor ruthenium red and the Ca2+efflux inhibitors Na' and Li+. These data indicated that the cyclic Ca2+ flux across the mitochondrial membrane was the primary event in the induction of mitochondrial swelling. In addition, the inhibition of swelling by a proton conductor CIPhzC(CN)2 suggested the involvement of proton fluxes in the mechanism of swelling. These observations led us to propose a tentative mechanism of action of unsaturated fatty acids which is illustrated in Fig.13. The unsaturated fatty acids and the saturated fatty acids of an appropriate chain length can dissolve in the membrane core and traverse it in the undissociated form. Ca2+ is taken up by the ruthenium-red-sensitive electrophoretic mechanism and is accumulated in the matrix space (Fig. 13A). On the inner surface of the membrane, Ca2+ binds with carboxyl groups of two molecules of fatty acids with the concomitant release of two H + ions into the matrix space-a reaction facilitated by high Ca2+ and low H + concentrations in the matrix (Fig. 13B). The Ca2+/fatty acid complex diffuses in the direction of a low Ca2+ concentration in the outer space (Fig. 13C). The accumulation of H + in the matrix induces a coupled H + / K + exchange [25]. The operation of such a cycle results in the net accumulation of K + ions in the matrix space. The entry of an anion (e.g. succinate) completes the conditions required for the osmotic swelling. The proposed mechanism is, in essence, an electroneutral Ca2+/H' exchange in which unsaturated fatty
Fatty-Acid-Induced Ca2+ Efflux from Mitochondria
622
acids (or medium-chain-length saturated fatty acids) act as mobile Ca2+ and H + carriers. The electroneutral C a 2 + / H + exchange was already postulated by other authors as a mechanism of the Ca2+ efflux from liver mitochondria [18,22]. The inhibition by Na+ of the Ca2+ efflux can conveniently by explained by a competition between the two ions for binding with fatty acids at the inner surface of the membrane which is readily permeable to Na' [23] (Fig.l2), although other mechanisms cannot be excluded at present. An alternative possibility is that the unsaturated fatty acids increase the fluidity of the mitochondrial membrane and thereby activate an endogenous Ca2 carrier which catalyzes the CaZ efflux. Indeed, we have observed that the spontaneous Ca2 efflux from kidney mitochondria was stimulated by the agents which increased the membrane fluidity (e.g. diethyl ether), and it was inhibited by the agents which decreased the membrane fluidity (e.g. MgC12) (data not shown). Also, the spontaneous Ca2+ efflux was strongly activated when the incubation temperature was raised above 23 -25 However, the interpretation of such results is difficult since most factors which increase the membrane fluidity, stimulate the mitochondrial phospholipase A as well [26]. Therefore, on the basis of Ca2+ transport measurements alone it cannot be decided whether the increased Ca2+efflux is due to the activation of endogenous ionophores or due to increased fatty acid concentration in the mitochondrial membranes resulting from phospholipase A2 stimulation. In addition, if fatty acids acted as mobile Ca2+ carriers, their effects on the Ca2+ transport would be expected to depend on the membrane fluidity. It may be noted, however, that the powerful inhibition of the spontaneous Ca2+ efflux from kidney mitochondria by albumin, even at high incubation temperatures, strongly suggest that the endogenous Ca2+ ionophores might be fatty acids or their derivatives (see also [ZS]). The observation that N a + ions inhibit the spontaneous Ca2 efflux from freshly prepared kidney mitochondria (Fig. 2) suggests that the endogenous fatty acids may indeed catalyse the Ca2+ efflux in vivo. It is the membrane phospholipids which are the most likely source of the mitochondrial Unsaturated fatty acids. Most phospholipids contain unsaturated fatty acids in position C2 of glycerol, and the mitochondria show considerable activity of phospholipase A2 which hydrolyses the phospholipids in C2 position [26,27]. The further fates of the split fatty acids, however, and the control of their level in the mitochondrial membranes, make as yet an almost completely obscure picture. The inhibition by sodium of the Ca2+ efflux from kidney mitochondria may have some physiological significance. In kidney cells the extrusion of Ca2+from +
+
+
I
+
T
.
the cell to the interstitial fluid proceeds via an Na'/ Ca2+ exchange mechanism: the Ca2+efflux from the cell is coupled to the Na+ influx into the cell [29]. The inhibition by Na+ of mitochondrial Ca2+efflux would provide a negative feedback loop between the Ca2 transport in the plasma membrane and in mitochondria. This is just the opposite to the positive feedback observed in the heart [15,16]. Such a mechanism is not unreasonable, however, if we realize that the kidney cells do transport large amounts of Ca2+ from the tubule lumen to the peritubular fluid. In this situation, the inhibition by sodium of mitochondrial Ca2+ efflux may be essential for keeping the cytosolic Ca2+ at low levels in face of varying loads of Ca2+ transported across the cell. +
This work was supported in part by grant 11. 1.1.5 from the Polish Academy of Sciences.
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I. Roman, P. Gmaj, C. Nowicka, and S. Angielski 22. Puskin, J. S., Gunter, T. E., Gunter, K. K. & Russel, P. R. (1 976) Biochemistry, 15, 3834- 3842. 23. Douglas, M. G. & Cockrell, R. S. (1974) J . Biol. Chem. 249, 5462 - 5471. 24. Wojtczak. L. (1974) FEBS Lett. 44, 25-30. 25. Massari, S. Cuc Azzone, G. F. (1970) Eur. J . Biochem. 12, 301309.
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I. Roman, Division of Neurology, RG-20, University of Washington, School of Medicine, Seattle, Washington, U.S.A. 98195 P. Gmaj*, C. Nowicka, and S. Angielski, Zaklad Biochemii Klinicznej, Instytut Patologii, Akademia Medyczna, PL-80-211 Gdansk, Poland
* To whom corrcspondence
should be addressed
